Lipopolysaccharide and lipotheicoic acid differentially modulate epididymal cytokine and chemokine profiles and sperm parameters in experimental acute epididymitis

Bacterial infections are the most prevalent etiological factors of epididymitis, a commonly diagnosed inflammatory disease in the investigation of male infertility factors. The influence of early pathogenic mechanisms at play during bacterial epididymitis on reproductive outcomes is little understood. We report here that experimental epididymitis induced in rats by Gram-negative (LPS) and Gram-positive (LTA) bacterial products resulted in differential patterns of acute inflammation in the cauda epididymis. LPS elicited a strong inflammatory reaction, as reflected by upregulation of levels of mRNA for seven inflammatory mediators (Il1b, Tnf, Il6, Ifng, Il10, Nos2 and Nfkbia), and tissue concentration of six cytokines/chemokines (IL1A, IL1B, IL6, IL10, CXCL2 and CCL2) within the first 24 h post-treatment. Conversely, LTA induced downregulation of one (Nfkbia) and upregulation of six (Il1b, Il6, Nos2, Il4 Il10 and Ptgs1) inflammatory gene transcripts, whereas increased the tissue concentration of three cytokines/chemokines (IL10, CXCL2 and CCL2). The stronger acute inflammatory response induced by LPS correlated with a reduction of epididymal sperm count and transit time that occurred at 1, 7, and 15 days post-treatment. Our study provides evidence that early epididymal inflammatory signaling events to bacterial activators of innate immunity may contribute to the detrimental effects of epididymitis upon male fertility.


Distribution of Blue Evans Dye into the epididymis after intravasal injection
Since the rat epididymis is highly segmented by connective tissue septa 1 , which could provide a physical barrier to ascending pathogens 2 , we initially performed experiments using saline solution containing Blue Evans tracer injected into the vas deferens to demonstrate its distribution into the cauda epididymis ( Supplementary Fig. S1). After 30 min, we observed intense blue staining in the distal region of the cauda epididymis (regions 18-19 of the rat epididymis 1 ), which became faint in the proximal cauda region (regions 15-17) and not detectable in any other proximal region of the organ ( Supplementary Fig. S1). These results demonstrated that the ascent of the dye was limited to the cauda epididymis, which is the major epididymal region affected by LPS-and LTA-induced inflammation in our experimental conditions.

Effects of LPS-and LTA-induced epididymitis on the morphology of the epididymis
We investigated the presence of histological signs of epididymal inflammation following 6 h and 24 h of 25 μg LPS or 125 μg LTA intravasal treatments. Epididymides from saline-control rats showed normal morphology with intact epithelial and interstitial compartments, and luminal

On the rationale for LPS and LTA dose selection
Because different LPS serotypes have different ability to induce inflammation, we selected the doses of ultrapure LPS based on its biological activity, measured as endotoxin units (EU) (1 ng ultrapure LPS from E. coli 055:B55 = 10 EU, as informed by the manufacturer, Innaxon, Cat# IAX-100-013). Thus, we employed ultrapure LPS doses equivalent to 50,000 (5 μg), 125,000 (12.5 μg) and 250,000 (25 μg) EU per epididymis. The highest dose we used was 4-8 fold lower than the LPS dose, in EU, used to induce epididymitis via endotoxin injection into the interstitial compartment of the rat caput epididymis 3,4 , indicating the sensitivity of our experimental model to trigger an acute inflammatory response in the rat epididymis. Since a similar rationale of dose-selection could not be used for LTA, we used LPS doses as reference to investigate its acute effects in the epididymis. Despite the difficulty to directly compare LPS and LTA, due to their different chemical and biological nature, we compared the effects of both PAMP treatments on the mRNA levels of the inflammatory markers Il1b and Tnf (used as readouts) to establish their optimal dose to induce inflammation in the cauda epididymis. Our results showed that only Il1b mRNA levels could be used as a readout, since LTA did not change the expression of Tnf transcripts at any dose analyzed. We could not explore highest LTA doses due to limitations on the volume of solution that could be injected into the vas deferens. Nevertheless, our approach allowed us to determine optimal LPS (25 µg) and LTA (125 µg) doses capable to induce an inflammatory reaction in the cauda epididymis at comparable levels based on Il1b mRNA changes. The presence of similar macroscopic and histological displays of inflammation in both LPS-and LTA-treated rats at the same time-point further confirmed an ongoing acute epididymitis.

Supplementary Table S1
Concentration of cytokines and chemokines in the cauda epididymis from saline-control, LPS-and LTA-treated rats. Data represent mean ± SEM of experiments performed with samples from the indicated number of rats.

Cytokine/Chemokine
Each sample was normalized to its respective total protein concentration and expressed as μg/mg of protein.
Asterisks indicate statistically different from the respective saline-control group (ANOVA followed by Bonferroni test, p<0.05)

Supplementary Table S2
Effect of intravasal LPS or LTA injection on kinematics parameters of spermatozoa from the cauda epididymis from 7-day saline-(control), LPS-and LTA-treated rats.